Expression And Function Of Nerve Growth Factor And Its Receptor In Reproductive System In Cow

It is now clear that nerve growth factor(NGF), a number of a family of proteins known as the neurotrophins(NTs), which is not only important for the differentiation and survival of neuronal cells, but is also required for the differentiation and proliferative activity of nonneuronal cells , including the cardiovascular, immune, endocrine, and reproductive systems.Recent studies showed that NGF acting via TrkA receptor is importantly involved in the control of early ovarian development, ovaluation, synthesis of ovarian hormone and ovarian diseases. NGF may facilitate cyclic development of uterus. NGF in the oviduct may be important in the transport of early zygote, fertilization and early embryonic development.The adult cows were used to investigate:1) the expression and location of the NGF and TrkA in ovary, uterus and oviducts. 2) the effects of gonadotropic hormone on the expression of NGF and TrkA in oviduct epithelial cells. 3) the effects of NGF on the expression of gonadotropins receptors in oviduct epithelial cells. The study would establish a foundation for demonstrating the biological roles and mechanism of NGF and TrkA in female reproduction. The principal results were described as following:Firstly, we investigated the localization and the expression of NGF and TrkA in the ovaries, uteri and oviducts during the follicular phase and the luteal phase with fluorescence quantitative-PCR, Western Blotting, and Immunofluorescence. The results showed that NGF and its receptor TrkA were expressed in the follicular cells of preantral follicle, and in granulosa cells and inner thecal cells of antral follicle during the follicular phase. In luteal phase ovary, NGF and its receptor TrkA were expressed in the follicular cells and the lutein cells. Furthermore, immunolocation of NGF and TrkA were expressed in the endometrial epithelial cells and oviduct epithelial cells during the follicular phase and the luteal phase. The amount of NGF mRNA in uterus was significantly higher than that in ovary during the follicular phase(P<0.05). The amount of TrkA mRNA in oviduct was significantly higher than that in ovary from the follicular phase (P<0.01). The expression level of NGF and TrkA mRNA in other tissues from the luteal phase was not significant difference. The amount of NGF mRNA in ovary, uterus or oviduct from two stages changed, but no significant difference. The presence of NGF and TrkA was demnonstrated in the reproductive organs, demonstrating that NGF and TrkA have important autocrine and paracrine regulatory in gonadal function.Secondly, we investigated the effects of gonadotropin on the expression of NGF and TrkA in oviduct epithelial cells with fluorescence quantitative-PCR and ELISA.The results of fluorescence quantitative-PCR indicated that the expression level of NGF mRNA were significantly higher than those in the control when treated with LH (10 or 20μg/L) (P<0.05); although the level of TrkA mRNA increased , no statistically difference was observed (P>0.05). The expression level of NGF mRNA in oviduct epithelial cells cultured with FSH at the concentration of 20 ug/L were significantly higher than those in the control(P<0.05), and the amount of TrkA mRNA also significantly increased when treat with FSH (10 or 20μg/L).The results of ELISA showed that the expression level of NGF protein increased in the LH and FSH groups than those in the control, but no statistical difference(P>0.05). These results showed that the gonadotropins could affect the expression of NGF and TrkA in oviduct epithelial cells.Thirdly, we investigated the effects of NGF on the expression of gonadotropin receptors in oviduct epithelial cells with fluorescence quantitative-PCR. The expression level of LHR mRNA in oviduct epithelial cells cultured media with NGF at the concentration of 30, 60 or 100 ug/L were significantly higher than those in the control(P<0.05).The expression level of FSHR mRNA in oviduct epithelial cells cultured media with NGF at the concentration of 30 or 60 ug/L were significantly higher than those in the control(P<0.05). The expression of LHR and FSHR decreased when oviduct epithelial cells were cultured media with K252α. These data showed that the NGF affected the expression of gonadotropin receptors in oviduct epithelial cells.Overall, our results indicated that NGF and TrkA were present in ovaries, uterus and oviducts of cow; the gonadotropins could enhance the expression of NGF and TrkA; the NGF and TrkA could enhance the expression of gonadotropin receptors.